Human cytomegalovirus (HCMV) is a beta-herpesvirus and is an important opportunistic pathogen of immunocompromised adults and can cause neurological abnormalities in some infants infected in utero (1). Such infected individuals are prone to suffer from a variety of diseases, including retinitis and systemic sequelae. The 230 kilobase (kb) genome of HCMV is similar in structure to that of herpes simplex virus (HSV), another human herpesvirus (2). HCMV has long and short unique regions, UL and US, respectively, each bounded by inverted repetitions. The UL region is bounded by the TRL (terminal repeat of the long) region and the IRL (inverted repeat of the long) region. The US region is bounded by the TRS (terminal repeat of the short) region and the IRS (inverted repeat of the short) region. Sequencing of the entire HCMV genome was completed recently (3). Sequencing indicates that the cytomegalovirus genome contains over 200 significant open reading frames. To date, relatively few of these open reading frames have been studied as to the function of the proteins which they may encode.
In the HSV system, the analysis of viral mutants has proven to be invaluable in the study of the function of many viral genes. Several temperature-sensitive mutants of HSV have been described. Other studies of HSV mutants have exploited the viral thymidine kinase gene as a selectable marker for site-directed mutagenesis (4-7). Random mutagenesis of HSV through the use of mini-Mu phage and transposon Tn5 has also been reported (8,9).
More recently, the prokaryotic .beta.-galactosidase gene has been used for site-directed insertion into the HSV genome, allowing for the easy identification of viral mutants as blue plaques after overlay with a chromogenic substrate, X-gal (10,11). In contrast to conditionally lethal mutants in which mutations lie in essential genes, these latter methodologies offer the advantage that mutants in non-essential genes can be created and identified. These successful strategies for the creation of HSV mutants have not been employed to any great extent in the HCMV system.
Probably due to the long replication cycle of this virus, there have only been a few reports concerning the creation or analysis of HCMV temperature-sensitive mutants. Due to the limited host range of HCMV in tissue culture (human diploid fibroblasts), mutagenesis by exploitation of a thymidine kinase gene would be exceedingly difficult.
To date, the only successful site-directed insertion of a marker gene into the genome of HCMV has been the insertion of .beta.-galactosidase within a repeat sequence region bounding UL (12).
However, genomic DNA analyses (12) of this recombinant indicated that an unexpected deletion of 5.5-kb of viral DNA sequences adjacent to the site of the insertion had occurred. One hypothesis given for this deletion was that the size of wild-type DNA is very close to the maximal size of packageable viral DNA, so that large insertions into the wild-type genome are not tolerated without a compensatory deletion elsewhere.
At present, there are few therapeutic agents approved for use against HCMV-infected patients. Ganciclovir, a nucleoside analog sold by Syntex Laboratories, Inc., under the tradename "CYTOVENE", is approved for use against HCMV-induced retinitis. However, ganciclovir is associated with side effects such as neutropenia and azoospermia. In addition, it has low oral bioavailability and thus must be administered intravenously.
Accordingly, there is a need for additional therapeutic agents against HCMV having decreased side effects and improved modes of administration.